BackgroundSemen hyperviscosity (SHV) is a condition that can markedly impair the physical and chemical characteristics of seminal fluid, which leads to an adverse impact on sperm function.The aim of this study was to assess the prevalence of SHV in infertile Egyptian men and to evaluate its effects on physical and biochemical semen parameters, and also to identify any correlation between SHV and infections or inflammation of the genital tract.Participants and methodsSemen samples were studied for 300 infertile men; of these, 225 men had normal semen viscosity and were excluded from the study. Seventy-five men with SHV were selected and included in this study. In addition, 25 fertile men with completely normal semen parameters who had recently fathered children were included as a control group. All participants underwent seminal analysis and measurement of seminal plasma fructose, ascorbic acid, calcium, and zinc, in addition to a peroxidase test and polymorphonuclear granulocyte elastase (PMN elastase).ResultsOur study showed that the prevalence of SHV was 25% in infertile Egyptian men. Sperm motility and sperm vitality, in addition to fructose, ascorbic acid, calcium, and zinc levels, were significantly reduced, whereas PMN elastase levels were significantly increased in samples with hyperviscosity (P < 0.05). There was a strong positive correlation between PMN elastase levels and increasing viscosity.ConclusionOur results showed that hyperviscosity seems to be the result of infection or inflammation in 75% in our cases and hyperviscosity seems not to be because of a single pathogenic factor, but rather because of several (biochemical, enzymatic, and genetic) factors that act in synergy. These factors should be studied further.

Semen is composed of fluids secreted by the male accessory glands, which contain proteins essential to the coagulation and liquefaction of semen. Hypofunction of the prostate or seminal vesicles causes abnormal viscosity of seminal fluid [1]. Seminal hyperviscosity is associated with increased oxidative stress in infertile men and increased proinflammatory interleukins in patients with male accessory gland infection, and more when the infection spreads to the seminal vesicles [2]. Semen hyperviscosity (SHV) seems to be associated with reduced sperm motility, possibly because of a trapping effect that prevents normal sperm progression through the female genital tract [3]. SHV also leads to certain technical difficulties in the handling of samples, such as when using Percoll gradients to prepare semen for in-vitro fertilization programs [4]. Physical and chemical methods have been proposed for the treatment of SHV. The physical method involves forcing the sample through a hypodermic needle [5], whereas the chemical treatment uses mucolytic agents such as a-chymotrypsin [6] and sputolysin [7]. However, one cannot ignore the fact that such procedures may damage the sperm structure. The pathogenic aspects of SHV are yet to be fully clarified. Gonzales [8] and Andrade-Rocha [9] reported reduced levels of fructose in SHV, and hypothesized inadequate functioning of the seminal vesicles as an explanation. Carpino and Siciliano [10] studied the possible correlation between SHV and protein secretion of the epididymis, seminal vesicles, and prostate. They found that hyperviscosity plays no role in the semen coagulation process. Mendeluk et al.[11] reported that there was no difference in the concentration of total proteins or DNA or in the percentage of water content in hyperviscous seminal plasma. In addition, Mendeluk et al.[12] observed that lysozyme plays no direct role in SHV, although a deficiency in cases of chronic infections could be an aggravating factor from a clinical standpoint.

Aim of the work

The aims of this study are to (a) determine the prevalence of SHV in infertile Egyptian men; (b) to assess the effects of SHV on physical and biochemical semen parameters; and (c) to identify any correlation between SHV and infections or inflammation of the genital tract.

Participants and methods

A prospective study was carried out during the period from June 2012 to April 2014 and included 300 infertile men who consecutively attended the infertility clinic at Damietta University Hospital, Al-Azhar University. A total of 225 men had normal semen viscosity and were excluded from the study. Seventy-five infertile men showed increased semen viscosity (study group) and were included in this study. In addition, 25 fertile men with completely normal semen parameters who had recently fathered children were included as a control group. The mean ages of the men in both infertile and control groups were 30.5 ± 3.5 (range 26-40) years and 31.5 ± 4.5 (range 25-41) years, respectively. This study was carried out in accordance with the guidelines of hospitals' ethics committee. All participants provided their informed consent for participation. An assessment of the full medical history was performed, and the participants underwent a diagnostic program comprising clinical and seminal analysis and measurement of biochemical markers of male accessory glands seminal plasma including fructose, ascorbic acid, calcium, and zinc. In addition, the peroxidase test was performed and polymorphonuclear granulocyte elastase (PMN elastase) seminal levels were assessed.

Laboratory evaluation

Semen collection and evaluation

All semen samples were obtained by masturbation after 3-5 days of abstinence. Samples were stored in a controlled incubator (37°C) on a gently moving plate. After liquefaction, semen analyses were carried out according to the recommendations in the WHO manual [13]. The specimens were analyzed for volume, pH, color, liquefaction time, viscosity, agglutination, sperm concentration, percent and grade of motility, sperm morphology, sperm vitality, total sperm count, and total motile sperm.

Semen viscosity evaluation

A measurement of viscosity was performed as recommended in the WHO manual [13], which defines an abnormal viscosity sample as one that can be drawn out to more than 2 cm with a rod or a pipette. Viscosity was determined 1 h after ejaculation by gently aspirating semen into a 5 ml pipette and then producing semen drops. In cases of abnormal viscosity, the drop formed a thread longer than 2 cm, which was evaluated using a centimeter scale. Hyperviscosity was graded under normal gravity as being mild (length of thread, >2 and ≤4 cm), moderate (>4 and ≤6 cm), or severe (>6 cm). According to semen viscosity, the participants were classified as follows: study group (infertile group with hyperviscosity) and control group (fertile group with normal viscosity).

Biochemical analysis

The seminal plasma was centrifuged at 3000 rpm for 15 min within 1 h after sampling and the supernatant was carefully removed and stored at -20°C for biochemical analysis of ascorbic acid, fructose, zinc, and calcium contents. Estimation of ascorbic acid was carried out using the 2,4-dinitrophenyl-hydrazine method: ascorbic acid in seminal plasma is oxidized by cupric (Cu 2+ ) ion to form dehydroascorbic acid, which reacts with acidic 2,4-dinitrophenyl-hydrazine to form a red bis-hydrazone, which was read on a spectrophotometer at 520 nm [14]. The normal adult male seminal reference values were 220-670 μmol/l. Serum zinc and calcium levels were measured using an atomic absorption spectrophotometer (model 4100; Perkin Elmer, Norwalk, Connecticut, USA). Fructose was measured using acid resorcinol colorimetry as described by Foreman [15]. The normal adult male seminal reference values were as follows: zinc: 72-183 mg/l, calcium: 450-672 mg/l, and fructose: 200-400 mg/dl.

Male genital tract infection and inflammation determination

Peroxidase test

Leukocytes were counted using the Leucoscreen method [16]. Briefly, one drop of semen was mixed with one drop of working solution (Leucoscreen stain and hydrogen peroxide), covered with a cover slip for 2 min, and then the result was read at a magnification of ×400. Peroxidase-positive cells stained yellow to brown, whereas other cells stained pink.

Polymorphonuclear granulocyte elastase

PMN elastase was measured using the homogeneous immunoassay Ecoline PMN elastase (Merck, Darmstadt, Germany). For this purpose, seminal plasma was mixed with latex particles coated with antibody fragments [F(ab´)2] against human PMN elastase, and after agglutination, the opacity proportional to the elastase concentration was measured photometrically using a standard curve and expressed in ng/ml. PMN elastase levels can be classified into three groups: less than 250 ng/ml (normal); 250-450 ng/ml (mild); 450-1000 ng/ml (moderate); and more than 1000 ng/ml (high inflammation) [17].

The data showed no statistically significant differences in the mean age in both groups. The mean ages of men in both infertile and control groups were (30.5 ± 3.5, range 26-40 years, and 31.5 ± 4.5, range 25-41 years, respectively; P > 0.05).

Prevalence of semen hyperviscosity

The results obtained in the present study showed that the prevalence of SHV was 25% (75/300 participants), with 20% with mild SHV, 40% with moderate SHV, and 40% with severe SHV as shown in [Figure 1].

Figure 1: Prevalence of semen hyperviscosity (SHV) in infertile Egyptian men. (1) Infertile men with normal viscosity. (2) Infertile men with hyperviscosity. The prevalence of SHV was 25% in infertile Egyptian men.

The present study showed that leukocytospermia was significantly increased in the study group (37.5%) compared with the control group (12.5%) (P < 0.05), as shown in [Table 1].

Correlation between hyperviscosity and biochemical parameters

The results obtained in the present study showed that seminal fructose levels and ascorbic acid were slightly but significantly lower in the study group compared with the control group (140 ± 20 vs. 220 ± 25 mg/dl and 383.13 ± 94.89 vs. 448.71 ± 98.13 μmol/l, respectively; P < 0.05). The seminal concentrations of zinc and calcium were significantly decreased (P < 0.05) in the study group compared with the control group (60 ± 15 vs. 88.5 ± 26.5 mg/l and 315 ± 25 vs. 520 ± 32 mg/l, respectively). The results (mean ± SD) are presented in [Table 2] and [Figure 2].

Figure 2: Data on biochemical parameters in the fertile group with normal viscosity and the infertile group with hyperviscosity. (1) Infertile group with hyperviscosity. (2) Fertile group with normal viscosity. Values with different scripts are significantly different between columns (fructose, ascorbic acid, calcium, and zinc levels) in both groups (P < 0.05, significant). There was a negative correlation between hyperviscosity and fructose, ascorbic acid, calcium, and zinc levels.

The results obtained in the present study showed that seminal PMN elastase levels were significantly increased (P < 0.05) in the study group compared with the control group. PMN elastase levels were mild (350 ± 30 ng/l) in 15 (20%) men with mild hyperviscosity, intermediate (670 ± 80 ng/l) in 45 (60%) men with moderate hyperviscosity, and high (1050 ± 50 ng/l) in 15 (20%) men with severe hyperviscosity, whereas in the control group, the PMN elastase levels were normal (150 ± 70 ng/l). The results (mean ± SD) are presented in [Table 3] and [Figure 3].

Figure 3: Polymorphonuclear granulocyte elastase (PMN elastase) levels in the control group and the study group. Values with different scripts are significantly different between columns (PMN elastase levels) in both groups (P < 0.05, Significant).

Our study showed that the prevalence of SHV was 25% in infertile Egyptian men. This prevalence was in agreement with that reported by Elia et al. [18] and was considerably lower than that found by Gonzales et al. [19], but considerably higher compared with that found by Gopalkrishnan et al. [20] and Esfandiari et al. [21]. This may be because of differences in patient populations, and may also be because of variations in the standardization of SHV measurement. Our study clearly found significant negative correlations between SHV and sperm motility, grade of motility, total motile sperm, and sperm vitality, in agreement with the studies carried out by Elzanaty et al. [3]. This study clearly showed significant negative correlations between SHV and seminal concentrations of fructose, ascorbic acid, zinc, and calcium, which is in agreement with the study carried out by Fernando and Rua [22]. Fructose and ascorbic acid are markers of seminal vesicle function, whereas calcium and zinc are markers for prostatic function so that physical analysis of ejaculate including viscosity was found to be clinically useful for the evaluation of the secretory activity of the seminal vesicles and prostate. Our results showed that PMN elastase concentrations were correlated negatively with sperm motility and vitality, which is in agreement with the study carried out by Kopa et al. [23]. Our results showed that PMN elastase levels were correlated negatively with ascorbic acid, fructose, zinc, and calcium concentrations. In addition, there was a positive correlation between PMN elastase levels and increasing viscosity, which is in agreement with the study carried out by Wolff et al. [24]. PMN elastase is secreted by activated granulocytes and has been shown to be a sensitive and quantitative marker of genital tract inflammation [18]. Our study clearly found a significant positive correlation between leukocytospermia and increasing viscosity. According to the WHO criteria, leukocytospermia was defined as the presence of more than 1 × 10 6 white blood cells/ml of semen and is considered a possible indicator of ongoing male genital tract infection [13]. Our results showed that leukocytospermia was found in 37.5% of the men in the infertile group with hyperviscosity, whereas PMN elastase levels was found in 75% in the same group. Our study showed that hyperviscosity seems to be a result of infection or inflammation in 75% in our cases. This infection or inflammation leads to changes in the biochemical components such as fructose, ascorbic acid, calcium, and zinc of seminal fluid, which in turn exerted negative effects on sperm motility and vitality, which may be lead to a potential impact on semen quality and fertilizing capacity of spermatozoa.

Our study clearly showed that PMN elastase is more useful, reliable, and suitable as a marker for silent genital tract inflammation and or infection than the peroxidase test, which depends on leukocyte concentrations. Moreover, the results of the study confirm the need for a change in the threshold value of peroxidase-positive cells according to the WHO definition to lower levels for the definition of silent genital tract inflammation.